Vaginal curettage as a means of diagnosing pregnancy in the guinea-pig.

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VAGINAL CURETTAGE AS A MEANS O F DIAGNOSING
PREGNAKCY IN THE GUINEA-PIG'
G . LOMBARD KELLY
Departnient of Anatomy, Medical Department, University of Georgia, Augusta
OKE TEXT FIGURE A N D ONE EIELIOTYPE PLATE
(FOUR FIGURES)
INTRODUCTION
The fact that the vaginal epithelium of the guinea-pig, like
that of several other rodents, undergoes a very marked transformation during pregnancy has been known f o r many years.
Lataste(4) seems to have been the first investigator to make
a note of this phenomenon ( '87), and under his direction
Xorau(5, 6) published two papers bearing directly on the
subject ( '88, '89). Following these writers, articles appeared
by Salvioli(8) and de Retterer(7), in 1890 and 1892. Since
then this subject has been treated extensively in the literature.
Recently, the present writer(1) has taken up the investigation and followed it from a number of angles, working out
the vaginal epithelial changes in the guinea-pig under a wide
variety of conditions. This paper cites most of the literature in this particular field up to the present.
As stated in this article, the changes that occur in the
vaginal epithelium are quite marked. The stratified squamous epithelium of estrus is transformed gradually to a high
columnar, mucous epithelium (figs. 2 and 3 ) . The writer
has shown (see reference 1) that this change begins about
'Contribution no. 3, series B, from the Department of Anatomy, Medical
Department of the University of Georgia. This work has been aided by a grant
from .the National Research Council through its Committee for Research on
Problems of Sex.
373
T H E ANATOMlC.41, RECORD, VOL. 40, NO. 3
374
G . LOMBARD KELLY
the second week of pregnancy and is well developed by the
end of the third week. From this time on the changes that
occur are due in large part to the hypertrophy of the vagina,
though the mueous nature of the cells becomes enhanced as
pregnancy continues.
Between estrous periods the vaginal mucosa assumes a
traiisitiorial form; it is very thin and cannot be describcd a s
strictly stratified syuamous or simple columnar. For illustrations of this form see reference l. Since no mucous
cells are present in this epithelium, curettage at this interval
will give negative scrapings.
METHODS
The extent of this epithelial transformation first came to
my attention while working on the mechanism of the opening
and closing of the vaginal orifice in the guinea-pig(2), a
female at term having been sacrificed during this investigation. It occurred to me then that, if these cells could be
scraped from the vagina, they would serve as a means of
diagnosing pregnaiicy in this animal. Some time later, I
happened t o be examining vaginal smears from guinea-pigs
daily, and in many of them I noticed the large mucous cells
that I have since found are present in large numbers after
abortions o r deliveries (figs. 4 and 5). Their presence in
such smears is due to their desquamation after the termination of the pregnant state. The appearance of the cells in
these two figures is the same as when they are obtained b y
the method described below, that is, by vaginal curettage.
These cells are characteristic in appearance, being x7er.Jlarge and reticulated, and having a small, dark nucleus. They
are as a whole basophilic and take a pale blue or lavender
stain. Usually they are seen in clumps, as shown in the
illustrations ; very seldom singly. By sectioning vaginas of
animals that had been pregnant from seven to nine weeks,
it was easy to identify these cells as the high columnar,
mucous epithelial cells of pregnancy (compare figs. 3 and 5).
DIAGNOSING PREGNANCY IN THE GUINEA-PIG
375
Since it has been found that very few of these mucous cells
desquamate before delivery, the vaginal smear as first described by Stockard and Papanicolaou(9) is not satisfactory
for obtaining cells from the vaginal wall as a diagnostic
method. It is true, however, that many smears from pregnant animals will show quite a number of these cells; but
all smears do not. Consequently, curettage seemed to be the
best avenue of approach.
Fig. 1
Thr rdurettr.
Some difficulty was experienced in devising a curette that
would work satisfactorily. The epithelium was so smooth
that the edge of the curette did not take hold easily, Finally,
the type illustrated (fig. 1) was found to accomplish the
purpose. It was made from a narrow, flat piece of steel by
bending one end back to less than a right angle with the shaft
and then sharpening the bent-over end to a fine edge.
The curettage is performed as follows: With the aid of
an assistaiit, the animal is wrapped in a towel with only its
376
G . LOMBARD KELLY
head out, in order to prevent struggling, and given just sufficient ether to get it asleep. The vulvar site is cleaned with
a sponge saturated with alcohol and the vulvar lips torn open.
The curette is now introduced and the curettage performed,
pressing the instrument firmly against the surface scraped
(usually the ventral wall of the vagina). With the end of
a clean pair of forceps the curetted material is pushed from
the angle of the curette to a glass slide and smeared there.
Fixation is with alcohol-ether, equal parts ; staining, with
liaematin and eosin. The entire procedure up to fixation
requires but a few minutes, and the animal is usually awake
by the time the slide is placed in the alcohol-ether.
In giving guinea-pigs ether it is advisable to use an indirect method, that is, to pump it from a bottle with a rubber
hulh. The gas is thus delivered to the animal’s nostrils
through a rubber tube, on the end of which the lialf of an
old rubber bulb with two or three air holes in its sides can
he attached as a nose-piece. If ether is applied directly, postoperative pneumonia is apt to be frequent. Incidentally, we
have never had any accidents whenever these methods have
beeii used.
OBSERVATIONS
At times it is very desirable t o know whether a female
guinea-pig is pregnant or not. The long gestation period
(nine weeks) tends to make this knowledge even more desirable if non-pregnant animals are needed within a short
time f o r experimental work.
On January 18, 1928, we received a shipment of ten female
guinea-pigs. These animals had been ordered for a particular
experiment that required non-gravid animals. On January
23rcl, vaginal curettage was undertaken, with the results
shomm in table 1.
As will be noted, two of these females had abortions the
day following arrival-a. not uncommon accident after long
transportation to new surroundings. Of course, the scrapings from these animals were positive, though they were
not then pregnant. A third animal was in stage 1 of estrns
377
DIAGNOSING PREGNANCY IX THE GUINEA-PIG
the day after arrival. Naturally, it had a negative scraping
three days later; but in examining the slides no identifying
marks revealed from which animal they came, each diagnosis
being made on the presence or absence of the characteristic
cells.
Since the vaginal epithelium of the high columnar, mucous
type does not develop until nearly three weeks after copulation, it can readily be seen that two curettages, three weeks
apart, are necessary in order to rule out pregnancy. A curetTABLE 1
This shows the results obtained in curetting a new shipment of ten animals
-
-.
NO.
-
1051
1052
1053l
1054
1055
1056a
1057
1058
1059'
*
.
.
~
FIRST CURETTAGE
L
SECOND CURETTAGE
~
FINAL DIAGNOSIS
CORRECT
--
~-
Yes
Yes
1/23/28, negative
1/23/28, doubtful
2/24/28, positive
2/14/28, positive
Positive
Positive
......
....
1/23/28,
1/23/28,
1/23/28,
1/23/28,
1/23/28,
None
None
Nolie
2/14/28, positive
None
Positive
Positive
Negative
Positive
Positive
Yes
Yes
Yes
Yes
Yes
......
positive
positive
negative
negative
positive
......
......
......
None
-~~
__
......
Negative
....
Yes
~
Animal 1060 returiied to estrus before time f o r the second curettage.
tage once a week for three weeks would be better. This
explains why three of the animals referred to were first
diagnosed as non-pregnant, though a subsequent examination
revealed positive findings. They had been pregnant less than
three weeks when they arrived.
It is worthy of note that, with this precaution, the final
diagnoses were all correct (omitting the two animals that
were known just to have aborted). I n doing this work, especial care was taken to avoid bias. No palpations were made
and none of the animals were big with young when they came.
Finally, the slides were stained and mounted by an assistant,
anct the eliamiiier did not know one slide from aiiotlier, but
hased his diagnosis only on the histological appearance.
In the spring of 1928, we were doing some experiments on
artificial insemination of the guinea-pig by way of the ovarian
hursa(3) ailti were very desirous of knowing as soon as possible whether a particular animal was impregnated or not.
Of course, those that returned to estrus a t the expected time
WIT
not pregnant, but about the others we could not be
absolutely sure, since these animals may occasionally have
estrus so weakly expressed that the vaginal orifice does not
opcn. So in each of this group a curettage was done three
weeks after the operation. Sever1 animals were operate(!
upon. Three came hack into estrus and the remaining four
were cnrctted. All four of the latter showed positive scrapi n g ~and subsequently were found actually to be pregnant.
Tn tr'ying out this method in tlie heginning a large nnmbcr'
of c u r d tages were done on animals whose state was kiiowii,
h u t the scrapings were examined without knowledge of their
source. It was soon found that it was not at all a difficult
mattor to tell wlietlicii* a given slide was from a pregnaiit or
11 oil-pregnaii t animal.
DISCUSSION
making such diagnoses three limitations must he boriie
miacl : First, the vagirial epithelium of immature females
is of the high columnar, mucous type and practically identical
with that of advanced pregnancy. Consequently, curettage
0 1 1 ariimals that have iiever come into estrus woalcl show
the mucous cells. The cells, howcver, arc smaller than those
of pregnancy, though the difference in size is not very great.
These animals are very small and their sexual immaturity
ciiii usually be inferred from their size. While the majority
of them have their first estrus at about three months of age,
we have had quite a number that were only a little over two
months old when the first stage-1 smear was obtained.
Secondly, curettage of animals within one or two weeks
after abortion or tldivery will probahly reveal mucous cells
111
iii
DIAGNOSING PREGNANCY I N T H E GUINEA-PIG
379
in the scrapings, since all the cells do not desquamate a t
the delivery estrus. Occasionally, the mucous cells may persist until the estrus following the delivery estrus, but not
thereafter in a scraping will they be abundant, as from a
pregnant animal, though in smears a few may be seen for
two or three weeks longer. I n a vaginal scraping from a
pregnant animal the domiiiancy of the mucous cells is easily
apparent.
A third limitation has already hecn mentioned, viz., the
animal must be pregnant three weeks before the test is
effective.
For those experienced enough to determine early pregnancy
in the guinea-pig by palpation, this method may not hold
much of value. It may provide a method of differential diagnosis in case of abdominal tumors, but this is doubtful. Still,
many persons can not by palpation of this aiiimaI determine
pregnancy until it is well advanced. For them vaginal curettage should solve the question. We have already found it
practicable within its limitations and often very useful. I t s
theoretical value is not without interest.
Finally, the question arises as to the diagnostic value of
this procedure in other species. We have not tried vaginal
curettage in other animals, with one exception, and can say
nothing about it. A student became interested in the method
and tried it on white rats known to be pregnant, but without
success. That is, he found no characteristic mucous cells.
This x7ork was not supervised and possibly the curettage
was not properly done. It requires firm pressure to scrape
the mucous cells off.
Of course, in animals with a persistent form of epithelium
throughout pregnancy this method would have no value. For
this reason it has no application in the human (with our
present knowledge of the histology of the human vagina).
380
G . LOMBARD KELLY
CONCLUSIONS
1. By vaginal curettage pregnancy can be diagnosed in
guinea-pigs three weeks or more after copulation.
2. Positive scrapings can be obtained from sexually immature females and from females within two or three weeks
after abortion or miscarriage; these are limitations of the
method.
3. The method has no valne, so f a r as can be told a t present, in animals that do not have a marked transition in the
vaginal epithelium during pregnancy, such as is seen in the
guinea-pig.
LITERATURE CITED
The histological transformations in the vaginal epithelium of
the guinea-pig. (To be published.)
KELLY,G. L., AND PAPANICOLAOU
1927 The mechanism of the periodical
opening and closing of the vaginal orifice in the guinea-pig. Am.
Jour. Anat., vol. 40, no. 2.
KELLY,G . L., Err AL. Artificial insemination by way of the ovarian bursa in
the guinea-pig. (To be published.)
LATASTE,F. 1887 Rccherches de xo66thique. Actes de la Soe. Linnienne
de Bordeaux, T. 40, pp. 317, 360, and 500.
MORAU, 13. 1888 Des transformations pgriodiques de l’epith6lium de la
muqueuse de quelques rongeurs. Comptes Rendus de Biol., Paris,
85, T. 5, p. 831.
1889 Des transformations epith6liales de la muqueuse du vagiii
de quelques rongeurs. Jour. de 1’Anat. et Phys., Paris, T. 2.5, pp.
277-297.
DE RETTEEER,ED. 1892 Sur la morphologie et l’bvolution de I’epith6lium
du vagin de mammif6res. Comptes Rendus, SOC. de Biol., P a r k ,
95, T. 4, pt. 2, pp. 101-107.
SALVIOLI,
I. 1890 Contribution B la physiologie de l’epith6lium. De la
structure de l’epith6lium vaginal de la lapine et des modifications
qu’il suhit pendant la gestation. Translated from Atti d. r. Acad.
d. Sv. di Torino, svi.
STOCKARD AND PAPANICOLAOU
1917 The existence of a typical oestrous cycle
in the guinea-pig with a description of its histological and physiological changes. Am. dour. Anat., vol. 22, no. 2.
1 KELLY,G. L.
2
3
4
5
6
7
8
9
PLATE
381
I’LSTM 1
EXPLANATION OF FIGURES
9 Low-pnww niicropliotograpl~of v a g i n a of a guinea-pig 11he weeks ylrgn;tnt,
~howingthe. high c.oluinnar rnuvous epithelium. X 130.
3 High-powrr view of the same. Comp;ire tliesc cells w i t h those in figure 3.
Soticr the diffrrence i n color and sizr of tho nurlri in the two figurt,s. X 360.
4 Ilow-powrr mierophotogrnph of mucous cells :IS they :tppe:ir in :t vugiiial
scraping. The collwtion in e l u s t t ~ sis characteristic. X 150.
?.
High-power view of the same. Compare the size of thc crlls with t h a t of
the war-by leiirocytes. Note that the nuclei appear to be undergoing caryolysis.
Thcs rrtieular structure of t h r cells is well shown here. X 360.
3IAGNOSING PREGXAKCY I N THE GUINEA-PIG
0. I,ODIBABD KBIiIiY
383
THE ANATOMICAL RRCORD, VOL. 40, NO. 3
PLATE 1